Experimental use of phenolic compounds
Phenol is an important chemical in biological research. It is used primarily for isolation and purification of DNA and RNA. Pure phenol is solid at room temperature. Almost all biochemical uses of phenol use a water saturated phenol solution, or a mixture of phenol in chloroform. The purity and pH of phenol solutions used in biochemical work is very important. Oxidized phenol can result in DNA damage, and cannot be used. The pH of phenol solutions dramatically changes the solubility of DNA. Phenol is effective at denaturing and precipitating most proteins, and is an effective means of purifying DNA or RNA from protein contaminants.
Water saturated phenol is typically shipped and stored with an upper layer of buffer, containing Tris-HCl at the appropriate pH. It is important to pipet from the lower layer, which is the layer containing phenol. Phenol chloroform solutions are stored similarly. Phenol is sometimes stored with an antioxidant to preserve its quality. It should be stored refrigerated at 4C.
Phenol chloroform is typically used along with a small amount of iso-amyl alcohol (IAA) in concentrations of 25:24:1. This mixture helps in avoiding foaming which sometimes occurs with pure phenol chloroform. The mixture is sometimes abbreviated PCI.
Phenol, phenol/chloroform, and chloroform are solvents for the plastics used in disposable pipets (but not pipet tips). You must use glass pipets for handling.
To purify DNA, water saturated phenol (pH 7.9) or phenol chloroform is added to approximately equal amounts of water or buffer containing DNA and vortexed. Brief centrifugation brings the phenol to the bottom, leaving purified DNA in the water solution. Concentrated salt solutions can be slightly denser than water saturated phenol (although not phenol chloroform) so care should be used in identifying the correct layer in these cases. Protein typically is evident as a white precipitate forming at the boundary between the phenol and water phases. The water (or buffer) layer is carefully removed and transfered to another tube. A second or third extraction may be useful in removing large amounts of protein. A final extraction with pure chloroform is recommended for removal of residual phenol from the purified DNA, which can otherwise interfere with spectroscopic quantification and downstream enzymatic reactions.
To purify RNA, water saturated phenol or phenol chloroform is used at a pH of 4.5. At this pH, DNA is soluble in the phenol phase, rather than the water phase, while RNA remains in the water phase. This allows separation of the RNA from the DNA in samples.
The pH of phenol solutions is difficult to measure directly, although the pH of the buffer solution above the phenol is a good indication. To measure the pH of the phenol directly, Ambion recommends mixing 2 ml of water saturated phenol with 5 ml of methanol and 13 ml water to form a uniform solution, which can be measured with a pH meter. For phenol/chloroform solutions, they recommend mixing 2 ml of phenol/chloroform with 8 ml methanol and 10 ml water. See the Ambion Tech Note.
Phenol is caustic and causes nasty chemical burns which are slow to heal. It is also a systemic poison which can be rapidly absorbed through the skin. Phenol is an effective local anaesthetic, and you may not be aware of burns immediately. Inhalation of phenol fumes is dangerous and can result in permanent loss of smell. It is one of the more dangerous chemicals in a typical biochemistry laboratory. First aid treatment involves removal of clothing, excess material, and swabbing of the area with glycerol or polyethylene glycol - 300. Responders should wear gloves to prevent their own injury. Water only may be effective in removing excess material, but may increase the rate of systemic uptake. Nitrile and latex gloves may not be effective, especially with phenol/chloroform, although they provide protection at least briefly. Seek immediate medical attention. Hunter recommends emergency treatment of small burns with isopropanol or polyethylene glycol in ethanol -- typically isopropanol is readily available and would be the first choice. See PMID 1416322.
History of experimental phenol research
- See "The Nazi Doctors: Medical Killing and the Psychology of Genocide" by Dr. Robert Jay Lifton: http://www.holocaust-history.org/lifton/LiftonT257.shtml